Hypomorphic mutations in the Rag1 gene are the main cause of Omenn syndrome (OS) in humans, a unique combined immunodeficiency associated with severe autoimmune manifestations. OS patients suffer from overwhelming infections accompanied with severe T-cell infiltrate and organ damage. The disease is rapidly fatal unless treated by allogeneic hematopoietic stem cell transplantation (HSCT), however the results of this procedure are less satisfactory when no matched donors are available. Based on the experience of HSCT from HLA-identical donors, it can be anticipated that a new treatment for Rag1 hypomorphic mutants based on bone marrow transplantation of genetically corrected highly purified HSCs would be highly beneficial. In order to design such therapeutic approach it is first crucial to: a) define discrete genomic elements for appropriate Rag1 gene expression in a context of a viral vector, b) characterize the Rag1 hypomorphic protein in vitro and in vivo and its ability to compete with the wild-type Rag1 molecule, and c) establish a syngeneic bone marrow transplantation model for the rescue of newly generated Rag1 hypomorphic mice, that are characterized by a severe impairment in T- and B-lymphocyte development, associated with peripheral expansion of activated T cells, as observed in humans with hypomorphic Rag defects. This proposal presents preliminary data showing the design of new lentiviral vectors for the expression of Rag1 and their use for transduction of highly purified HSCs, in addition to the initial characterization of a newly developed hypomorphic Rag1 mouse. The proposed research program will focus on three aspects of Rag1 hypomorphism, which will significantly accelerate the advancement of the clinical development of a lentiviral based therapy for Rag related combined immunodeficiency. Lentiviral vectors carrying various genomic elements driving Rag1 expression will be studied in vivo for their ability to induce specific and appropriate gene expression. The ability of wild-type Rag1 protein to induce functional rearrangement when competing with its hypomorphic counterpart will be studied using an in vitro rearrangement assay and in pre-B cells isolated from Rag1 hypomorphic mice. Finally, results from aims 1 and 2 of this proposal will serve as the basis for preclinical studies aimed at rescuing Rag1 hypomorphic mice using lentiviral transduction of HSCs. Overall, this study will be important to facilitate development of novel forms of treatment for combined immunodeficiency due to hypomorphic RAG gene defects, based on gene transfer. PUBLIC HEALTH RELEVANCE: Immunodeficiencies due to Rag1 mutations are responsible for considerable mortality and available treatments are still limited. This proposal establishes the basis for the development of a new therapeutic approach for Rag related immunodeficiencies based on correction of hematopoietic stem cells by gene transfer.